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library("devtools")
install_github("jw156605/SLICER")
Skipping install of 'SLICER' from a github remote, the SHA1 (cb1be8ac) has not changed since last install.
Use `force = TRUE` to force installation
library(Seurat)
library(readr)
library(tidyverse)
library(HGC)
library(latticeExtra)
library(lattice)
library(RColorBrewer)
library(clusterProfiler)
library(org.Mm.eg.db)
library(AnnotationDbi)
exp = read_csv("normalized_data.csv")
New names:
• `` -> `...1`
• `` -> `...151`
• `` -> `...240`
• `` -> `...289`
• `` -> `...352`
• `` -> `...391`
• `` -> `...394`
• `` -> `...450`
• `` -> `...462`
Rows: 14414 Columns: 463
── Column specification ────────────────────────────────────────────────────────────────────────────────────────────────
Delimiter: ","
chr (1): ...1
dbl (454): M1_A01, M1_A02, M1_A03, M1_A05, M1_A06, M1_A08, M1_A09, M1_A10, M1_B01, M1_B03, M1_B04, M1_B05, M1_B06, M...
lgl (8): ...151, ...240, ...289, ...352, ...391, ...394, ...450, ...462
ℹ Use `spec()` to retrieve the full column specification for this data.
ℹ Specify the column types or set `show_col_types = FALSE` to quiet this message.
exp = discard(exp,~all(is.na(.) |. == ""))
exp = distinct(exp, `...1`, .keep_all = TRUE)
# filtering out the reprogramming transcription factors
exp = filter(exp, !`...1`%in% c("Mef2c", "Gata4", "Tbx5"))
exp = column_to_rownames(exp, var="...1")
seurat.obj = CreateSeuratObject(counts = exp)
seurat.obj = NormalizeData(seurat.obj)
Performing log-normalization
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seurat.obj = FindVariableFeatures(seurat.obj, selection.method = "vst", nfeatures = 10000)
Calculating gene variances
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Calculating feature variances of standardized and clipped values
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all.genes = rownames(seurat.obj)
seurat.obj = ScaleData(seurat.obj, features = all.genes)
Centering and scaling data matrix
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seurat.obj = RunPCA(seurat.obj, features = VariableFeatures(seurat.obj))
PC_ 1
Positive: Ehd4, Acaa2, Dsp, Ldhb, Nid2, Ryr2, Myl4, Epb4.1, Smpx, Tnni3
Ttn, Tnnt2, Mgst3, Sort1, Ldb3, Jup, Mlf1, Baiap2l1, Rrad, Mgrn1
Fryl, Ppp1r3c, Fam134b, Fabp3, Cenpa, Usp28, Clu, Pcyt1a, Ddit4l, Cd24a
Negative: Fstl1, Bgn, Cd200, Col3a1, Rbp1, Tagln2, Fam198b, Postn, Col1a2, Tagln
Fbln2, Rbms3, Lox, Fbn1, Loxl1, Dhrs3, Marcks, Col8a1, 11-Sep, Col5a2
Clca1, Lrrc8a, Spry2, Anxa1, Ogn, Nars, Gstm2, Fbln5, Asns, Acta2
PC_ 2
Positive: Top2a, Prc1, Smc2, Ccnb1, Birc5, Cdk1, Ube2c, Rrm2, Mki67, Pbk
Ckap2l, Asf1b, Cks2, Tpx2, Cdca8, Kif20b, 2810417H13Rik, Cks1b, Anln, Tacc3
Spc25, Hmmr, Hmgb2, Shcbp1, Ndc80, Mad2l1, Nuf2, Cenpe, Ect2, Cdca2
Negative: Cst3, B2m, Sptbn1, Malat1, Ypel5, Itm2b, Gyg, Parp3, Tspyl1, Serpine2
Lama2, Igfbp7, Ogn, B4galt1, Gpx3, Ctsa, Asah1, Pdcd4, Itgb5, Fhl2
Pam, Col4a2, Lhfp, Tcn2, Mxra8, Smim14, Ctsd, Akap13, Prex2, Impact
PC_ 3
Positive: Cst3, Col4a1, Flna, Igfbp7, Col1a1, Col4a2, Myl9, Sorbs2, Fhl2, Itgb1
Tns1, Col14a1, Ankrd1, Tpm2, Lbh, Pcolce, Tuba1a, Postn, Tuba1b, Itga1
Col5a1, Actn4, Pdlim2, Myom1, Pdgfrb, Tbx20, Sorbs1, Acta2, Pcdh18, Macf1
Negative: Gpm6a, Mgst1, Hpgd, Gsta4, Ptgs1, Upk1b, Pkhd1l1, F11r, Msln, Bst1
Maf, Ezr, Mpzl2, Cxadr, Krt8, Cd55, Igfbp6, Igfbp4, Acsl4, Bex4
Gcnt1, Vnn1, Ftl1, Efna5, Slpi, Ptp4a2, Tmem108, Tmem154, Csde1, Bnc1
PC_ 4
Positive: S100a6, Tpm1, Tpm4, Actb, Nme1, Zyx, Lgals1, Tpm2, Cryab, Actg1
Wisp1, Gapdh, Gm12070, Actn1, Timp1, Slc25a5, Sec13, Eif5a, Psat1, Creb3l1
Tnfrsf12a, Ccnd2, Csf1, Pkm, Pcolce, Acta2, Des, Rcn3, Col1a1, Wdr1
Negative: Egfl7, Ctla2a, Gja4, Emcn, Cdh5, Notch1, Klhl4, Gimap6, Pecam1, Stab1
Gpr116, Lyl1, Tie1, Entpd1, Ctla2b, Fli1, Myct1, Sox7, Thsd1, Eltd1
Gngt2, Mmrn2, S1pr1, Mecom, Tgfbi, Klhl24, Tmem2, Apoe, Plxnd1, Akap13
PC_ 5
Positive: Egfl7, Notch1, Klhl4, Stab1, Cdh5, Emcn, Cgnl1, Pecam1, Gja4, Ramp2
Lyl1, Col4a1, Gpr116, Tie1, Entpd1, Zfp69, Loxl2, Eltd1, Plxnd1, Thsd1
Cxcl16, Col5a1, Tek, S1pr1, Txndc5, Tgfbi, Odc1, Eng, Sox7, Myct1
Negative: Spcs3, Impact, Ypel5, Cpne8, Lnp, Arhgap44, Sptbn1, Myo10, Nexn, Arid5b
Arl5a, Braf, Klhl9, Cpne3, Lama2, Mrfap1, Serping1, Mlf1, Zdhhc2, Mier3
Gm2a, Dstn, Emc2, Myoz2, Ncam1, Rhobtb3, Cryab, Gucy1a3, Ttn, Idh1
ElbowPlot(seurat.obj)
seurat.obj = FindNeighbors(seurat.obj, dims = 1:15)
Computing nearest neighbor graph
Computing SNN
seurat.obj = FindClusters(seurat.obj, resolution = 1.75)
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 454
Number of edges: 16805
Running Louvain algorithm...
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Maximum modularity in 10 random starts: 0.4797
Number of communities: 8
Elapsed time: 0 seconds
identified_cells = read.csv("paper_cell_states.csv", row.names = 1, header=TRUE)
identified_cells = identified_cells[match(rownames(seurat.obj@meta.data), rownames(identified_cells)),]
seurat.obj@meta.data$cc_paper = identified_cells$CC
seurat.obj@meta.data$cat_paper = identified_cells$stage
seurat.obj@meta.data$cc_hclust = "CCI"
seurat.obj@meta.data[active_cells,]$cc_hclust = "CCA"
Idents(seurat.obj) = "RNA_snn_res.1.75"
seurat.obj = RunUMAP(seurat.obj, dims = 1:15)
Warning: The default method for RunUMAP has changed from calling Python UMAP via reticulate to the R-native UWOT using the cosine metric
To use Python UMAP via reticulate, set umap.method to 'umap-learn' and metric to 'correlation'
This message will be shown once per session
17:59:25 UMAP embedding parameters a = 0.9922 b = 1.112
17:59:25 Read 454 rows and found 15 numeric columns
17:59:25 Using Annoy for neighbor search, n_neighbors = 30
17:59:25 Building Annoy index with metric = cosine, n_trees = 50
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17:59:25 Writing NN index file to temp file /var/folders/fj/09brpj8s5mz1c5wxktl80s080000gn/T//RtmpE0XJwn/file8ed51e4226b8
17:59:25 Searching Annoy index using 1 thread, search_k = 3000
17:59:25 Annoy recall = 100%
17:59:26 Commencing smooth kNN distance calibration using 1 thread with target n_neighbors = 30
17:59:27 Initializing from normalized Laplacian + noise (using irlba)
17:59:27 Commencing optimization for 500 epochs, with 17028 positive edges
Using method 'umap'
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17:59:28 Optimization finished
clusters = DimPlot(seurat.obj, group.by = "RNA_snn_res.1.75", label = TRUE, reduction = "umap")
condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE, reduction = "umap")
condition|clusters
seurat.obj = RunTSNE(seurat.obj, dims = 1:15)
DimPlot(seurat.obj, reduction = "tsne", group.by = 'RNA_snn_res.1.75')
markers = FindAllMarkers(seurat.obj, logfc.threshold = 0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')
top_markers = markers %>%
group_by(cluster) %>%
slice_max(n = 2, order_by = avg_log2FC)
FeaturePlot(seurat.obj, features = c("Dhrs3"), min.cutoff = 'q10')
VlnPlot(seurat.obj, features = c("Rbp1", "Dhrs3"))
VlnPlot(seurat.obj, features = c("Fstl1", "Bgn"))
VlnPlot(seurat.obj, features = c("Serpinh1", "Dsp"))
VlnPlot(seurat.obj, features = c("Prc1", "Birc5"))
## cell cycle
s.genes <- str_to_title(cc.genes$s.genes)
g2m.genes <- str_to_title(cc.genes$g2m.genes)
seurat.obj = CellCycleScoring(seurat.obj, s.features = s.genes, g2m.features = g2m.genes)
clusters = DimPlot(seurat.obj, group.by = "RNA_snn_res.1.75", label = TRUE, reduction = "umap")
real_condition = DimPlot(seurat.obj, group.by = "cc_paper", label = TRUE, reduction = "umap")
phase = DimPlot(seurat.obj, group.by = "Phase", label = TRUE, reduction = "umap")
cat = DimPlot(seurat.obj, group.by = "cc_hclust", label = TRUE, reduction = "umap")
clusters|real_condition|phase|cat
NA
NA
subset(x = seurat.obj, subset = cc_hclust == "CCA")
An object of class Seurat
14410 features across 80 samples within 1 assay
Active assay: RNA (14410 features, 10000 variable features)
3 dimensional reductions calculated: pca, umap, tsne
cci_seurat = FindNeighbors(cci_seurat, dims = 1:15)
cci_seurat = FindClusters(cci_seurat, resolution = 1)
cl= DimPlot(cci_seurat, group.by = "RNA_snn_res.1",label = TRUE)
pap = DimPlot(cci_seurat, group.by = "cat_paper", label = TRUE)
cl|pap
cci_markers = FindAllMarkers(cci_seurat, logfc.threshold = 0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')
cci_top_markers = cci_markers %>%
group_by(cluster) %>%
slice_min(n = 5, order_by = p_val_adj)
VlnPlot(cci_seurat, features = c("Fstl1", "Bgn"))
VlnPlot(cci_seurat, features = c("Tnni3", "Smpx"))
VlnPlot(cci_seurat, features = c("Serpinh1", "Dsp"))
VlnPlot(cci_seurat, features = c("Clca2", "Rbp1"))
VlnPlot(cci_seurat, features = c("Cfh", "B4galt1"))
Fstl1 involved in endothelial cell differentiation and endothelial cell migration and Bgn is a gene that is used for connective tissue metabolism -> lower expression -> cluster 0 = iCM
Tnni3, smpx, dsp used for cardiomycoytes -> cluster 2 = pre-iCM, cluster 1 and 3 are still fibroblasts
cluseter 1 further away from cluster 0 = fibroblastr clusetr 3 = induced fibroblasts
Idents(cci_seurat) = "RNA_snn_res.1"
cci_seurat = RenameIdents(cci_seurat, `0` = "iCM")
cci_seurat = RenameIdents(cci_seurat, `1` = "Fib")
cci_seurat = RenameIdents(cci_seurat, `2` = "piCM")
cci_seurat = RenameIdents(cci_seurat, `3` = "iFib")
cca_seurat = FindClusters(cca_seurat, resolution = 1)
Modularity Optimizer version 1.3.0 by Ludo Waltman and Nees Jan van Eck
Number of nodes: 80
Number of edges: 2244
Running Louvain algorithm...
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Maximum modularity in 10 random starts: 0.2948
Number of communities: 3
Elapsed time: 0 seconds
cl= DimPlot(cca_seurat, group.by = "RNA_snn_res.1",label = TRUE)
pap = DimPlot(cca_seurat, group.by = "cat_paper", label = TRUE)
cl|pap
cca_markers = FindAllMarkers(cca_seurat, logfc.threshold = 0.25, min.pct = 0.1, only.pos = FALSE, test.use = 'MAST')
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 1
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Done!
Combining coefficients and standard errors
Calculating log-fold changes
Calculating likelihood ratio tests
Refitting on reduced model...
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Done!
Calculating cluster 2
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Combining coefficients and standard errors
Calculating log-fold changes
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Refitting on reduced model...
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Done!
cca_top_markers = cca_markers %>%
group_by(cluster) %>%
slice_min(n = 5, order_by = p_val_adj)
VlnPlot(cca_seurat, features = c("Dab2", "Kcnab1"))
VlnPlot(cca_seurat, features = c("Cenpf", "Pttg1"))
VlnPlot(cca_seurat, features = c("Upk1b", "Timp3"))
NA
NA
dab2 involved in endothelial stuf -> 0 is probably induced cardiomyocyet Upk1b is involved in epithelial stuff -> 2 is porbably fibroblasts 1 - induced fibroblasts
Idents(cca_seurat) = "RNA_snn_res.1"
cca_seurat = RenameIdents(cca_seurat, `0` = "iCM")
cca_seurat = RenameIdents(cca_seurat, `1` = "iFib")
cca_seurat = RenameIdents(cca_seurat, `2` = "Fib")
DimPlot(cca_seurat, label = TRUE)
seurat.sc = CreateSeuratObject(counts = exp)
seurat.sc = SCTransform(seurat.sc)
seurat.sc = RunPCA(seurat.sc, features = VariableFeatures(seurat.sc))
identified_cells = identified_cells[match(rownames(seurat.sc@meta.data), rownames(identified_cells)),]
seurat.sc@meta.data$cc_paper = identified_cells$CC
seurat.sc@meta.data$cat_paper = identified_cells$stage
seurat.sc = RunUMAP(seurat.sc, dims = 1:15)
seurat.sc = FindNeighbors(seurat.sc, dims = 1:15)
seurat.sc = FindClusters(seurat.sc, resolution = 1.75)
seurat.sc = CellCycleScoring(seurat.sc, s.features = s.genes, g2m.features = g2m.genes)
clusters = DimPlot(seurat.sc, group.by = "SCT_snn_res.1.75", label = TRUE, reduction = "umap")
real_condition = DimPlot(seurat.sc, group.by = "cc_paper", label = TRUE, reduction = "umap")
phase = DimPlot(seurat.sc, group.by = "Phase", label = TRUE, reduction = "umap")
cat = DimPlot(seurat.sc, group.by = "cat_paper", label = TRUE, reduction = "umap")
clusters|real_condition|phase|cat